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Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive

  • Submitted25-02-2026|

  • Accepted30-03-2026|

  • First Online 11-04-2026|

  • doi 10.18805/LRF-939

ABSTRACT

Background: Yardlong bean [Vigna unguiculata (L.) Walp. subsp. sesquipedalis] is a significant vegetable legume in Southeast Asia, where breeding programs focus on pod length, yield and uniformity. Prolonged selection for uniform commercial traits may progressively reduce genetic variability within hybrid germplasm if not systematically monitored.

Methods: Twenty commercial F1 yardlong bean hybrids were evaluated using seventeen morphological traits. Multivariate analyses (correlation, UPGMA and principal component analysis) were performed to assess phenotypic structure. Genetic diversity was examined using nuclear ITS, chloroplast psbA-trnH and RAPD markers.

Result: Morphological differentiation was moderate and primarily driven by pod-related quantitative traits, particularly pod length and pod weight. DNA barcoding confirmed species identity with complete sequence conservation among hybrids. RAPD analysis revealed substantial polymorphism (79.42%) and genetic similarity coefficients ranging from 0.46 to 0.96, indicating detectable genetic variation among hybrids. However, RAPD is a dominant marker system with limited reproducibility; therefore, validation using co-dominant markers, such as SSR or SNP, is recommended. Hybrids Y15, Y16 and Y18 combined superior pod traits with high genetic similarity, while Y12 and Y20 were genetically distinct. The results indicate that phenotypic similarity does not necessarily reflect genomic similarity. A combined analytical framework integrating phenotypic evaluation with molecular data provides a more reliable basis for germplasm characterization and breeding decisions.

INTRODUCTION

Yardlong bean [Vigna unguiculata (L.) Walp. subsp. Sesquipedalis] is widely cultivated throughout tropical and subtropical agroecosystems, where it serves as an important vegetable crop in both subsistence and market-oriented production systems. In Southeast Asia, it represents an important vegetable crop with strong market demand, where consumer preferences strongly favor pod length, tenderness and visual quality (Widyawan et al., 2021).
       
Yield variation in yardlong bean is primarily associated with pod-related quantitative traits, particularly pod length and pod weight, which are consistently prioritized in breeding programs targeting market acceptance (Tantasawat et al., 2010; Widyawan et al., 2021). Genetic studies have demonstrated that both additive and non-additive gene effects contribute to the inheritance of yield-related traits in cowpea, highlighting their importance in breeding programs (Singh et al., 2016).
       
Conserved genomic regions, such as ITS and psbA-trnH, are frequently used for species-level identification because their sequences are relatively stable across taxa (Kress et al., 2005; Stefanović et al., 2009). In contrast, RAPD markers are effective for detecting genome-wide polymorphism and have been widely applied in cowpea and yardlong bean diversity studies (Pidigam et al., 2019; Widyawan et al., 2021).
       
The incorporation of molecular markers alongside morphological traits enables a more comprehensive assessment of genetic structure and diversity within breeding materials. Recent advances in cowpea genetics and breeding highlight the importance of integrating phenotypic and molecular approaches for sustainable crop improvement (Kim et al., 2025). Therefore, this study aimed to (i) evaluate phenotypic variation among commercial hybrids, (ii) assess genetic diversity using multiple marker systems and (iii) examine the relationship between phenotypic and molecular patterns for breeding utilization.

MATERIALS AND METHODS

Plant materials and experiment design
 
Twenty commercial F1 hybrids (labeled Y1-Y20) were sourced from seed companies in Vietnam, Thailand and China (Table 1). The experiment was conducted from January 2025 to May 2025 using a completely randomized design with three replications at the Institute of Biotechnology, Hue University, Vietnam. Each plant was grown individually under consistent conditions to minimize environmental variation.

Table 1: List of commercial F1 hybrids of yardlong bean (V. unguiculata) used in this study.


 
Morphological analysis
 
Seventeen traits were evaluated using standard descriptors (Bioversity International, 2007), including 10 qualitative and 7 quantitative characters. Quantitative traits included leaf length, leaf width, petiole length, pod length, pod diameter, pod weight and Brix.
       
Data were analyzed using correlation analysis, UPGMA clustering and principal component analysis (PCA). All multivariate analyses were conducted using PAST software version 4.03 (Hammer et al., 2001).
 
Molecular analysis
 
DNA extraction and sequencing analysis
 
Genomic DNA was extracted using a CTAB-based protocol (Truong et al., 2024). PCR amplification and sequencing were performed following standard procedures. Sequence alignment and phylogenetic analysis were conducted in MEGA 12 (Kumar et al., 2024) using the Neighbor-Joining method with 1000 bootstrap replicates. The sequences related to this study were submitted to the NCBI and are available at https://submit.ncbi.nlm.nih.gov/subs/?search=SUB16004427.
 
ITS and trnH-psbA analysis
 
PCR amplification was performed using the ITSu1-ITSu4 primer pair following the protocol described by Rasphone et al. (2022). Additionally, variation in chloroplast DNA was assessed with the trnH–psbA intergenic spacer (Vir et al., 2023).
 
RAPD analysis
 
A preliminary RAPD screening was conducted using genomic DNA from two representative accessions (Y2 and Y12) and a set of 100 UBC (Bioneer, Korea) RAPD primers (Supplementary Table 1). To validate the reproducibility and applicability of the selected primers, two additional accessions (Y14 and Y15) were subsequently included, allowing assessment of polymorphism across the broader germplasm set and providing a more reliable estimation of genetic diversity. PCR amplification conditions followed the protocol described by Truong et al. (2013).

Supplementary Table 1: List of RAPD primers used for primary screened in this study.


       
Marker parameters, including PB, PPB, PIC, MI, Rp and EMR, as well as genetic diversity indices (Na, Ne, h, I), were calculated. Cluster analysis was conducted using UPGMA in PAST software (Hammer et al., 2001).

RESULTS AND DISCUSSION

Morphological characterization
 
Morphological variation among the evaluated hybrids was moderate, with differentiation largely associated with pod-related traits rather than vegetative characteristics (Table 2, Table 3; Fig 1, Fig 2). Leaf size varied moderately, while petiole length exhibited a narrow range, indicating weaker selection pressure on vegetative traits (Widyawan et al., 2021). In contrast, pod traits displayed substantial variation, with pod length (26.18-40.19 cm), diameter (0.74-0.88 cm) and weight (11.11-14.17 g) contributing most to phenotypic diversity and yield (Table 3), consistent with previous findings in vegetable cowpea (Sathish et al., 2023). Similar studies have also highlighted the importance of pod diameter, number of pods per plant and yield-related traits as key selection criteria in cowpea improvement programs (Acharya et al., 2025). Brix values (4.92-7.08%) indicated variation in quality traits (Choi et al., 2024). Correlation analysis indicated strong positive relationships among several pod traits, particularly between pod length and pod weight. The absence of intraspecific variation indicates a high level of sequence conservation within the evaluated material (Fig 3), whereas Brix showed weak relationships, suggesting partial independence between yield and quality traits (Sathish et al., 2023; Choi et al., 2024). UPGMA clustering grouped hybrids into three clusters (Fig 4), primarily driven by pod traits. At the same time, PCA confirmed their dominant role in variation (Fig 5). Overall, phenotypic variation is moderate and largely governed by pod-related traits.

Table 2: Qualitative morphological characterization of twenty F1 commercial hybrids of V. unguiculata.



Table 3: Quantitative morphological characterization of F1 commercial hybrids of V. unguiculata.



Fig 1: Variation in leaflet morphology among twenty F1 commercial hybrids (Y1-Y20) of yardlong bean (V. unguiculata).



Fig 2: Representative pods showing differences in pod color and length between yardlong bean F1 commercial hybrids Y10 (upper) and Y11 (lower).



Fig 3: Ellipse-based correlation plot showing relationships among quantitative morphological and quality traits in yardlong bean F1 commercial hybrids (V. unguiculata) corresponding to quantitative traits (Table 3).



Fig 4: UPGMA dendrogram showing phenotypic relationships among twenty yardlong bean (V. unguiculata) F1 commercial hybrids based on morphological traits.



Fig 5: PCA biplot showing the relationships between morphological traits and the distribution of twenty yardlong bean (V. unguiculata) F1 commercial hybrids along the first two principal components.


 
DNA barcoding analysis (ITS and psbA-trnH)
 
All twenty F1 yardlong bean hybrids were successfully sequenced for ITS (666 bp) and psbA–trnH (334 bp) regions, showing 100% query coverage and identity with V. unguiculata reference sequences, with no polymorphic sites detected (Table 4). Phylogenetic analysis revealed that all hybrids clustered with V. unguiculata references with strong bootstrap support (100%) in both ITS and psbA-trnH trees (Fig 6; Fig 7), clearly separating them from other Vigna species and confirming their taxonomic identity.

Table 4: Sequencing performance and GenBank similarity of ITS and psbA-trnH regions in 20 F1 commercial hybrids of V. unguiculata.



Fig 6: Phylogenetic relationships of 20 F1 commercial hybrids of V. unguiculata inferred from ITS sequences.



Fig 7: Phylogenetic relationships of 20 F1 commercial hybrids of V. unguiculata based on psbA-trnH sequences.


       
The absence of intraspecific variation indicates strong sequence conservation, which is common in domesticated crops due to founder effects and selection pressure (Doebley et al., 2006; Olsen and Wendel, 2013). Similar low variation in barcode regions has been reported in legumes (Stefanović et al., 2009). Among available DNA barcodes, ITS has been preferentially applied due to high interspecific divergence (Baldwin et al., 1995), while psbA-trnH is an effective chloroplast marker for distinguishing taxa (Kress et al., 2005; Shaw et al., 2005; Shaw et al., 2007). The congruent phylogenetic patterns confirm species identity but indicate limited resolution for intraspecific diversity.
 
RAPD analysis
 
RAPD markers detected considerable levels of genome-wide polymorphism among the hybrids, indicating the presence of measurable genetic diversity, consistent with previous reports in cowpea under different agro-climatic conditions (Kumar et al., 2017; Pidigam et al., 2019; Widyawan et al., 2021). Of 100 primers screened, eight produced clear and reproducible polymorphic patterns (Table 5; Fig 8). These generated 4-15 bands per primer (mean 9.50), with 79.42% polymorphism, consistent with previous studies (Pidigam et al., 2019; Saha et al., 2020). PIC values (0.15-0.30) and resolving power (1.0-4.3) indicated moderate marker informativeness (Botstein et al., 1980), while MI and EMR confirmed marker efficiency (Widyawan et al., 2021; Shubha et al., 2022).

Table 5: Polymorphic RAPD primers obtained from the initial screening were used for subsequent genetic diversity analyses of twenty V. unguiculata F1 commercial hybrids.



Fig 8: PCR-RAPD amplification profiles of twenty V. unguiculata F1 commercial hybrids generated using primer UBC#486 and UBC#487.


       
Genetic similarity ranged from 0.46 to 0.96 (Table 6), reflecting both closely related and divergent genotypes. UPGMA clustering grouped hybrids into four clusters (Fig 9), indicating moderate differentiation despite a shared commercial background. Genetic diversity indices (Na = 1.8289; h = 0.2669; I = 0.3999) further supported this pattern (Table 7).

Fig 9: UPGMA dendrogram illustrating the genetic relationships among twenty F1 commercial hybrids of V. unguiculata based on RAPD marker analysis.



Table 6: Pairwise genetic similarity coefficients among twenty F1 commercial hybrids of V. unguiculata.



Table 7: Genetic diversity indices of twenty F1 commercial hybrids of V. unguiculata revealed by RAPD analysis.


       
Comparison with morphology showed both concordance and incongruence. Hybrids Y15, Y16 and Y18 clustered consistently across analyses (Fig 4, Fig 5, Fig 9), while others showed divergence, indicating convergent selection (Sathish et al., 2023). DNA barcoding showed no variation (Fig 6; Fig 7), confirming species identity but limited diversity (Doebley et al., 2006). Overall, RAPD analysis revealed genetic variation not apparent from morphological observations alone.
 
Concordance and incongruence between phenotypic and molecular patterns
 
The integration of morphological traits, DNA barcoding and RAPD markers highlighted both agreement and discrepancy between phenotypic traits and molecular data. DNA barcoding revealed complete sequence conservation in the ITS and psbA-trnH regions, with 100% identity to V. unguiculata, confirming species identity but indicating limited variation at these loci. The lack of detectable polymorphism in these regions is consistent with patterns reported in domesticated crops, where historical bottlenecks and intensive selection have constrained variation in conserved loci (Doebley et al., 2006; Olsen and Wendel, 2013).
       
In contrast, RAPD analysis detected moderate polymorphism (79.42%) and genetic differentiation among hybrids, consistent with previous studies in yardlong bean and cowpea (Pidigam et al., 2019; Widyawan et al., 2021). Concordance was observed in hybrids Y15, Y16 and Y18, which showed both phenotypic similarity and high genetic relatedness, reflecting selection for pod-related traits such as pod length and weight (Sathish et al., 2023). These findings highlight the importance of integrating multiple marker systems to capture both conserved and variable genomic regions.
       
However, incongruence was evident in hybrids such as Y1 and Y3, which were morphologically similar but genetically distinct, suggesting convergent selection acting on different genetic backgrounds. The distinct position of Y12 further confirmed the presence of divergent genetic materials. These findings suggest that similar phenotypic expression does not necessarily correspond to underlying genetic similarity. Thus, integrating morphological evaluation with molecular data offers a more robust approach for germplasm characterization and breeding applications.
       
These results inform breeding strategies. Hybrids Y15, Y16 and Y18 are suitable elite parents for yield improvement but offer limited variability. In contrast, divergent genotypes such as Y12 provide valuable sources for broadening the genetic base. Crosses between elite and divergent lines are recommended to enhance variation and exploit heterosis (Boukar et al., 2019; Kim et al., 2025).

CONCLUSION

This study provides an integrated analysis of phenotypic and molecular variation in commercial yardlong bean hybrids. Morphological analysis revealed moderate variation mainly governed by pod-related traits, particularly pod length and weight, reflecting strong yield-oriented selection. DNA barcoding confirmed species identity with complete sequence conservation, whereas RAPD markers detected moderate polymorphism and grouped hybrids into distinct clusters, indicating underlying genetic differentiation despite phenotypic similarity.
       
Some hybrids with similar pod performance were genetically distinct, while others showed both phenotypic and molecular similarity, demonstrating that phenotypic uniformity should not be interpreted as evidence of genetic uniformity. Hybrids Y15, Y16 and Y18 combined superior yield traits with high genetic similarity, making them suitable for production. Hybrid Y20 showed potential for quality improvement, while divergent genotypes such as Y12 provide valuable resources for broadening the genetic base. The combined use of phenotypic and molecular analyses offers a more robust framework for breeding-oriented germplasm evaluation.

ACKNOWLEDGEMENT

The present study was supported by the Core Research Program, Hue University (Grant No. NCTB.DHH.2024.03). We thank Dr. Sonexay Rasphone from Savannakhet University, Laos, for sharing plant materials.
 
Disclaimers
 
The authors are solely responsible for the content’s accuracy; the opinions expressed do not necessarily reflect those of their institutions and no liability is assumed for any consequences resulting from its use.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

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    • Submitted25-02-2026|

    • Accepted30-03-2026|

    • First Online 11-04-2026|

    • doi 10.18805/LRF-939

    ABSTRACT

    Background: Yardlong bean [Vigna unguiculata (L.) Walp. subsp. sesquipedalis] is a significant vegetable legume in Southeast Asia, where breeding programs focus on pod length, yield and uniformity. Prolonged selection for uniform commercial traits may progressively reduce genetic variability within hybrid germplasm if not systematically monitored.

    Methods: Twenty commercial F1 yardlong bean hybrids were evaluated using seventeen morphological traits. Multivariate analyses (correlation, UPGMA and principal component analysis) were performed to assess phenotypic structure. Genetic diversity was examined using nuclear ITS, chloroplast psbA-trnH and RAPD markers.

    Result: Morphological differentiation was moderate and primarily driven by pod-related quantitative traits, particularly pod length and pod weight. DNA barcoding confirmed species identity with complete sequence conservation among hybrids. RAPD analysis revealed substantial polymorphism (79.42%) and genetic similarity coefficients ranging from 0.46 to 0.96, indicating detectable genetic variation among hybrids. However, RAPD is a dominant marker system with limited reproducibility; therefore, validation using co-dominant markers, such as SSR or SNP, is recommended. Hybrids Y15, Y16 and Y18 combined superior pod traits with high genetic similarity, while Y12 and Y20 were genetically distinct. The results indicate that phenotypic similarity does not necessarily reflect genomic similarity. A combined analytical framework integrating phenotypic evaluation with molecular data provides a more reliable basis for germplasm characterization and breeding decisions.

    INTRODUCTION

    Yardlong bean [Vigna unguiculata (L.) Walp. subsp. Sesquipedalis] is widely cultivated throughout tropical and subtropical agroecosystems, where it serves as an important vegetable crop in both subsistence and market-oriented production systems. In Southeast Asia, it represents an important vegetable crop with strong market demand, where consumer preferences strongly favor pod length, tenderness and visual quality (Widyawan et al., 2021).
           
    Yield variation in yardlong bean is primarily associated with pod-related quantitative traits, particularly pod length and pod weight, which are consistently prioritized in breeding programs targeting market acceptance (Tantasawat et al., 2010; Widyawan et al., 2021). Genetic studies have demonstrated that both additive and non-additive gene effects contribute to the inheritance of yield-related traits in cowpea, highlighting their importance in breeding programs (Singh et al., 2016).
           
    Conserved genomic regions, such as ITS and psbA-trnH, are frequently used for species-level identification because their sequences are relatively stable across taxa (Kress et al., 2005; Stefanović et al., 2009). In contrast, RAPD markers are effective for detecting genome-wide polymorphism and have been widely applied in cowpea and yardlong bean diversity studies (Pidigam et al., 2019; Widyawan et al., 2021).
           
    The incorporation of molecular markers alongside morphological traits enables a more comprehensive assessment of genetic structure and diversity within breeding materials. Recent advances in cowpea genetics and breeding highlight the importance of integrating phenotypic and molecular approaches for sustainable crop improvement (Kim et al., 2025). Therefore, this study aimed to (i) evaluate phenotypic variation among commercial hybrids, (ii) assess genetic diversity using multiple marker systems and (iii) examine the relationship between phenotypic and molecular patterns for breeding utilization.

    MATERIALS AND METHODS

    Plant materials and experiment design
     
    Twenty commercial F1 hybrids (labeled Y1-Y20) were sourced from seed companies in Vietnam, Thailand and China (Table 1). The experiment was conducted from January 2025 to May 2025 using a completely randomized design with three replications at the Institute of Biotechnology, Hue University, Vietnam. Each plant was grown individually under consistent conditions to minimize environmental variation.

    Table 1: List of commercial F1 hybrids of yardlong bean (V. unguiculata) used in this study.


     
    Morphological analysis
     
    Seventeen traits were evaluated using standard descriptors (Bioversity International, 2007), including 10 qualitative and 7 quantitative characters. Quantitative traits included leaf length, leaf width, petiole length, pod length, pod diameter, pod weight and Brix.
           
    Data were analyzed using correlation analysis, UPGMA clustering and principal component analysis (PCA). All multivariate analyses were conducted using PAST software version 4.03 (Hammer et al., 2001).
     
    Molecular analysis
     
    DNA extraction and sequencing analysis
     
    Genomic DNA was extracted using a CTAB-based protocol (Truong et al., 2024). PCR amplification and sequencing were performed following standard procedures. Sequence alignment and phylogenetic analysis were conducted in MEGA 12 (Kumar et al., 2024) using the Neighbor-Joining method with 1000 bootstrap replicates. The sequences related to this study were submitted to the NCBI and are available at https://submit.ncbi.nlm.nih.gov/subs/?search=SUB16004427.
     
    ITS and trnH-psbA analysis
     
    PCR amplification was performed using the ITSu1-ITSu4 primer pair following the protocol described by Rasphone et al. (2022). Additionally, variation in chloroplast DNA was assessed with the trnH–psbA intergenic spacer (Vir et al., 2023).
     
    RAPD analysis
     
    A preliminary RAPD screening was conducted using genomic DNA from two representative accessions (Y2 and Y12) and a set of 100 UBC (Bioneer, Korea) RAPD primers (Supplementary Table 1). To validate the reproducibility and applicability of the selected primers, two additional accessions (Y14 and Y15) were subsequently included, allowing assessment of polymorphism across the broader germplasm set and providing a more reliable estimation of genetic diversity. PCR amplification conditions followed the protocol described by Truong et al. (2013).

    Supplementary Table 1: List of RAPD primers used for primary screened in this study.


           
    Marker parameters, including PB, PPB, PIC, MI, Rp and EMR, as well as genetic diversity indices (Na, Ne, h, I), were calculated. Cluster analysis was conducted using UPGMA in PAST software (Hammer et al., 2001).

    RESULTS AND DISCUSSION

    Morphological characterization
     
    Morphological variation among the evaluated hybrids was moderate, with differentiation largely associated with pod-related traits rather than vegetative characteristics (Table 2, Table 3; Fig 1, Fig 2). Leaf size varied moderately, while petiole length exhibited a narrow range, indicating weaker selection pressure on vegetative traits (Widyawan et al., 2021). In contrast, pod traits displayed substantial variation, with pod length (26.18-40.19 cm), diameter (0.74-0.88 cm) and weight (11.11-14.17 g) contributing most to phenotypic diversity and yield (Table 3), consistent with previous findings in vegetable cowpea (Sathish et al., 2023). Similar studies have also highlighted the importance of pod diameter, number of pods per plant and yield-related traits as key selection criteria in cowpea improvement programs (Acharya et al., 2025). Brix values (4.92-7.08%) indicated variation in quality traits (Choi et al., 2024). Correlation analysis indicated strong positive relationships among several pod traits, particularly between pod length and pod weight. The absence of intraspecific variation indicates a high level of sequence conservation within the evaluated material (Fig 3), whereas Brix showed weak relationships, suggesting partial independence between yield and quality traits (Sathish et al., 2023; Choi et al., 2024). UPGMA clustering grouped hybrids into three clusters (Fig 4), primarily driven by pod traits. At the same time, PCA confirmed their dominant role in variation (Fig 5). Overall, phenotypic variation is moderate and largely governed by pod-related traits.

    Table 2: Qualitative morphological characterization of twenty F1 commercial hybrids of V. unguiculata.



    Table 3: Quantitative morphological characterization of F1 commercial hybrids of V. unguiculata.



    Fig 1: Variation in leaflet morphology among twenty F1 commercial hybrids (Y1-Y20) of yardlong bean (V. unguiculata).



    Fig 2: Representative pods showing differences in pod color and length between yardlong bean F1 commercial hybrids Y10 (upper) and Y11 (lower).



    Fig 3: Ellipse-based correlation plot showing relationships among quantitative morphological and quality traits in yardlong bean F1 commercial hybrids (V. unguiculata) corresponding to quantitative traits (Table 3).



    Fig 4: UPGMA dendrogram showing phenotypic relationships among twenty yardlong bean (V. unguiculata) F1 commercial hybrids based on morphological traits.



    Fig 5: PCA biplot showing the relationships between morphological traits and the distribution of twenty yardlong bean (V. unguiculata) F1 commercial hybrids along the first two principal components.


     
    DNA barcoding analysis (ITS and psbA-trnH)
     
    All twenty F1 yardlong bean hybrids were successfully sequenced for ITS (666 bp) and psbA–trnH (334 bp) regions, showing 100% query coverage and identity with V. unguiculata reference sequences, with no polymorphic sites detected (Table 4). Phylogenetic analysis revealed that all hybrids clustered with V. unguiculata references with strong bootstrap support (100%) in both ITS and psbA-trnH trees (Fig 6; Fig 7), clearly separating them from other Vigna species and confirming their taxonomic identity.

    Table 4: Sequencing performance and GenBank similarity of ITS and psbA-trnH regions in 20 F1 commercial hybrids of V. unguiculata.



    Fig 6: Phylogenetic relationships of 20 F1 commercial hybrids of V. unguiculata inferred from ITS sequences.



    Fig 7: Phylogenetic relationships of 20 F1 commercial hybrids of V. unguiculata based on psbA-trnH sequences.


           
    The absence of intraspecific variation indicates strong sequence conservation, which is common in domesticated crops due to founder effects and selection pressure (Doebley et al., 2006; Olsen and Wendel, 2013). Similar low variation in barcode regions has been reported in legumes (Stefanović et al., 2009). Among available DNA barcodes, ITS has been preferentially applied due to high interspecific divergence (Baldwin et al., 1995), while psbA-trnH is an effective chloroplast marker for distinguishing taxa (Kress et al., 2005; Shaw et al., 2005; Shaw et al., 2007). The congruent phylogenetic patterns confirm species identity but indicate limited resolution for intraspecific diversity.
     
    RAPD analysis
     
    RAPD markers detected considerable levels of genome-wide polymorphism among the hybrids, indicating the presence of measurable genetic diversity, consistent with previous reports in cowpea under different agro-climatic conditions (Kumar et al., 2017; Pidigam et al., 2019; Widyawan et al., 2021). Of 100 primers screened, eight produced clear and reproducible polymorphic patterns (Table 5; Fig 8). These generated 4-15 bands per primer (mean 9.50), with 79.42% polymorphism, consistent with previous studies (Pidigam et al., 2019; Saha et al., 2020). PIC values (0.15-0.30) and resolving power (1.0-4.3) indicated moderate marker informativeness (Botstein et al., 1980), while MI and EMR confirmed marker efficiency (Widyawan et al., 2021; Shubha et al., 2022).

    Table 5: Polymorphic RAPD primers obtained from the initial screening were used for subsequent genetic diversity analyses of twenty V. unguiculata F1 commercial hybrids.



    Fig 8: PCR-RAPD amplification profiles of twenty V. unguiculata F1 commercial hybrids generated using primer UBC#486 and UBC#487.


           
    Genetic similarity ranged from 0.46 to 0.96 (Table 6), reflecting both closely related and divergent genotypes. UPGMA clustering grouped hybrids into four clusters (Fig 9), indicating moderate differentiation despite a shared commercial background. Genetic diversity indices (Na = 1.8289; h = 0.2669; I = 0.3999) further supported this pattern (Table 7).

    Fig 9: UPGMA dendrogram illustrating the genetic relationships among twenty F1 commercial hybrids of V. unguiculata based on RAPD marker analysis.



    Table 6: Pairwise genetic similarity coefficients among twenty F1 commercial hybrids of V. unguiculata.



    Table 7: Genetic diversity indices of twenty F1 commercial hybrids of V. unguiculata revealed by RAPD analysis.


           
    Comparison with morphology showed both concordance and incongruence. Hybrids Y15, Y16 and Y18 clustered consistently across analyses (Fig 4, Fig 5, Fig 9), while others showed divergence, indicating convergent selection (Sathish et al., 2023). DNA barcoding showed no variation (Fig 6; Fig 7), confirming species identity but limited diversity (Doebley et al., 2006). Overall, RAPD analysis revealed genetic variation not apparent from morphological observations alone.
     
    Concordance and incongruence between phenotypic and molecular patterns
     
    The integration of morphological traits, DNA barcoding and RAPD markers highlighted both agreement and discrepancy between phenotypic traits and molecular data. DNA barcoding revealed complete sequence conservation in the ITS and psbA-trnH regions, with 100% identity to V. unguiculata, confirming species identity but indicating limited variation at these loci. The lack of detectable polymorphism in these regions is consistent with patterns reported in domesticated crops, where historical bottlenecks and intensive selection have constrained variation in conserved loci (Doebley et al., 2006; Olsen and Wendel, 2013).
           
    In contrast, RAPD analysis detected moderate polymorphism (79.42%) and genetic differentiation among hybrids, consistent with previous studies in yardlong bean and cowpea (Pidigam et al., 2019; Widyawan et al., 2021). Concordance was observed in hybrids Y15, Y16 and Y18, which showed both phenotypic similarity and high genetic relatedness, reflecting selection for pod-related traits such as pod length and weight (Sathish et al., 2023). These findings highlight the importance of integrating multiple marker systems to capture both conserved and variable genomic regions.
           
    However, incongruence was evident in hybrids such as Y1 and Y3, which were morphologically similar but genetically distinct, suggesting convergent selection acting on different genetic backgrounds. The distinct position of Y12 further confirmed the presence of divergent genetic materials. These findings suggest that similar phenotypic expression does not necessarily correspond to underlying genetic similarity. Thus, integrating morphological evaluation with molecular data offers a more robust approach for germplasm characterization and breeding applications.
           
    These results inform breeding strategies. Hybrids Y15, Y16 and Y18 are suitable elite parents for yield improvement but offer limited variability. In contrast, divergent genotypes such as Y12 provide valuable sources for broadening the genetic base. Crosses between elite and divergent lines are recommended to enhance variation and exploit heterosis (Boukar et al., 2019; Kim et al., 2025).

    CONCLUSION

    This study provides an integrated analysis of phenotypic and molecular variation in commercial yardlong bean hybrids. Morphological analysis revealed moderate variation mainly governed by pod-related traits, particularly pod length and weight, reflecting strong yield-oriented selection. DNA barcoding confirmed species identity with complete sequence conservation, whereas RAPD markers detected moderate polymorphism and grouped hybrids into distinct clusters, indicating underlying genetic differentiation despite phenotypic similarity.
           
    Some hybrids with similar pod performance were genetically distinct, while others showed both phenotypic and molecular similarity, demonstrating that phenotypic uniformity should not be interpreted as evidence of genetic uniformity. Hybrids Y15, Y16 and Y18 combined superior yield traits with high genetic similarity, making them suitable for production. Hybrid Y20 showed potential for quality improvement, while divergent genotypes such as Y12 provide valuable resources for broadening the genetic base. The combined use of phenotypic and molecular analyses offers a more robust framework for breeding-oriented germplasm evaluation.

    ACKNOWLEDGEMENT

    The present study was supported by the Core Research Program, Hue University (Grant No. NCTB.DHH.2024.03). We thank Dr. Sonexay Rasphone from Savannakhet University, Laos, for sharing plant materials.
     
    Disclaimers
     
    The authors are solely responsible for the content’s accuracy; the opinions expressed do not necessarily reflect those of their institutions and no liability is assumed for any consequences resulting from its use.

    CONFLICT OF INTEREST

    The authors declare no conflicts of interest.

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